Inducible Nitric Oxide Synthase (iNOS) converts L-arginine to L-citrulline and NO using two molecules of oxygen and a tetrahydrobiopterin cofactor. There are several known physiological functions of NO generated by iNOS and its two constitutive isofrms (nNOS and eNOS) including regulation of vascular tone, neurotransmission, cell signaling, and combating infection. Several chronic inflammatory diseases including atherosclerosis, sepsis, cardiogenic shock, rheumatoid arthritis, inflammatory bowel disease, and transplant rejection all involve over-expression or increased iNOS activity. Over-expression of iNOS can lead to depletion of its substrate and cofactor resulting in the production of superoxide and other reactive oxygen species, which contribute to the pathophysiology of several chronic inflammatory disorders. Therefore, iNOS must be carefully regulated. Based on preliminary studies, carbon monoxide, which is produced physiologically by heme-oxygenase-1 (HO), directly inhibits the activity of iNOS through a reversible heme-ligand switching mechanism. Indoleamine 2,3 dioxygenase (IDO), which is responsible for the degradation of tryptophan, directly regulates the expression and activity of iNOS via tryptophan metabolites. Using spectroscopic techniques, such as resonance Raman scattering, the regulatory mechanisms involving the crosstalk of HO and IDO and iNOS will be studied. In addition, mutants will be studied to understand the structure of the catalytic intermediates and the oxygen chemistry of iNOS to serve as a basis for the development of mechanism-based inhibitors. Ultimately, this project will contribute to the understanding of how iNOS functions and is regulated in vivo. Knowledge gained from this research project will aid in the design of isoform specific inhibitors aimed at ameliorating the oxidative stress incurred in chronic inflammatory conditions.